Pattern formation is the activity by which embryonic cells form ordered spatial arrangements of differentiated tissues. The physical complexity of higher organisms arises during embryogenesis through the interplay of cell-intrinsic lineage and cell-extrinsic signaling. Inductive interactions are essential to embryonic patterning in vertebrate development from the earliest establishment of the body plan, to the patterning of the organ systems, to the generation of diversive cell types during tissue differentiation (Davidson, E., (1990) Development 108: 365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al., (1992) Cell 68: 257-270). The effects of developmental cell interactions are varied. Typically, responding cells are diverted from one route of cell differentiation to another by inducing cells that differ from both the uninduced and induced states of the responding cells (inductions). Sometimes cells induce their neighbors to differentiate like themselves (homoiogenetic induction); in other cases a cell inhibits its neighbors from differentiating like itself. Cell interactions in early development may be sequential, such that an initial induction between two cell types leads to a progressive amplification of diversity. Moreover, inductive interactions occur not only in embryos, but in adult cells as well, and can act to establish and maintain morphogenetic patterns as well as induce differentiation (J.B. Gurdon (1992) Cell 68:185-199).
Many types of communication take place among animal cells during embryogenesis, as well as in the maintenance of tissue in adult animals. These vary from long-range effects, such as those of rather stable hormones circulating in the blood and acting on any cells in the body that possess the appropriate receptors, however distant they are, to the fleeting effects of very unstable neurotransmitters operating over distances of only a few microns. Of particular importance in development is the class of cell interactions referred to above as embryonic induction; this includes influences operating between adjacent cells or in some cases over greater than 10 cell diameters (Saxen et al. (1989) Int J Dev Biol 33:21-48; and Gurdon et al. (1987) Development 99:285-306). Embryonic induction is defined as in interaction between one (inducing) and another (responding) tissue or cell, as a result of which the responding cells undergo a change in the direction of differentiation. This interaction is often considered one of the most important mechanism in vertebrate development leading to differences between cells and to the organization of cells into tissues and organs.
Receptor tyrosine kinases are apparently involved in many different process including cellular differentiation, proliferation, embryonic development and, in some cases, neoplastic growth. High affinity binding of specfic soluble or matrix-associated growth factor ligands can cause the activated receptor to associate with a specific repertoire of cytoplasmic singnalling molecules that can lead to a cascade of intracellular signalling resulting in, for example, activation or inactivation of cellular gene programs involved in differentiation and/or growth. Accordingly, peptide growth factors that are ligands for such receptor tyrosine kinases are excellent candidates for intercellular signaling molecules with important developmental roles. Indeed, these ligands are known to have potent effects on a wide variety of cell activities in vitro, including survival, proliferation, differentiation, adhesion, migration and axon guidance. The powerful signaling effects of these molecules are further emphasized by the ability of both the ligands and the receptors, when activated by mutation or overexpression, to become potent oncogenes and cause drastic cellular transformation (reviewed by Cantley et al. (1991) Cell 64:281-302; Schlessinger and Ullrich (1992) Neuron 9:383-391; and Fantl et al. (1993)Annu Rev Biochem 62:453-481).
To illustrate, specific developmental roles have been demonstrated for some growth factors or their tyrosine kinase receptors. For example, the c-kit receptor tyrosine kinase, encoded at the mouse W locus (Chabot et al. (1988) Nature 335:88-89; and Geissler et al. (1988) Cell 55:185-192) and its ligand KL, encoded at the mouse SI locus (Flanagan and Leder (1990) Cell 63:185-194; Copeland et al. (1990) Cell 63:175-183; Huang et al. (1990) Cell 63:225-233; and Zsebo et al. (1990) Cell 63:213-224), determine the proliferation, survival, and/or migration of primordial germ cells, hematopoietic stem cells, and neural crest progenitor cells. Other examples are the trk family ligands and receptors, with highly specific functions in the developing mammalian nervous system (Klein et al. (1993) Cell 75:113-122; and Jones et al. (1994) Cell 76:989-999) and the FGF receptor, implicated in Xenopus mesoderm induction (Amaya et al. (1991) Cell 66:257-270). In invertebrates, too, receptor tyrosine kinases and ligands such as sevenless, boss, torso, breathless and let-23 are known to play key roles in processes that range from setting up the primary embryonic axes to specifying the fate of a single cell in the ommatidium (Greenwald and Rubin (1992) Cell 68:271-281; Shilo (1992) Faseb J 6:2915-2922; and Zipursky et al. (1992) Cold Spring Harbor Symp Quant Biol 57:381-389). Taken together, the emerging picture of the developmental functions of receptor tyrosine kinases and their ligands is striking in that these molecules play key roles at all stages of embryonic development, and in a remarkable range of different types of patterning process.
The receptor tyrosine kinases can be divided into families based on structural homology and, in at least some cases, obvious shared functional characteristics (Fantl et al. (1993) Annu Rev Biochem 62:453481). The family with by far the largest number of known members is the EPH family. Since the description of the prototype, the EPH receptor (Hirai et al. (1987) Science 238:1717-1720), sequences have been reported for at least ten members of this family, not counting apparently orthologous receptors found in more than one species. Additional partial sequences, and the rate at which new members are still being reported, suggest the family is even larger (Maisonpierre et al. (1993) Oncogene 8:3277-3288; Andres et al. (1994) Oncogene 9:1461-1467; Henkemeyer et al. (1994) Oncogene 9:1001-1014; Ruiz et al. (1994) Mech Dev 46:87-100; Xu et al. (1994) Development 120:287-299; Zhou et al. (1994) J Neurosci Res 37:129-143; and references in Tuzi and Gullick (1994) Br J Cancer 69:417-421). Remarkably, despite the large number of members in the EPH family, all of these molecules were identified as orphan receptors without known ligands.
The present invention relates to the discovery of a novel EPH receptor ligand, referred to hereinafter asxe2x80x9cElf-1xe2x80x9d, which protein has apparently broad involvement in the formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, and can be used to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.
In general, the invention features an Elf-1 polypeptide, preferably a substantially pure preparation of an Elf-1 polypeptide, or a recombinant Elf-1 polypeptide. In preferred embodiments the polypeptide has a biological activity associated with its binding to an EPH receptor, e.g., it retains the ability to bind to a hek-related or sek-related receptor, though it may be able to either agnoize or antagonize signal transduction by the EPH receptor. The polypeptide can be identical to the mammalian Elf-1 polypeptide (muElf-1) shown in SEQ ID No: 2, or it can merely be homologous to that sequence. Likewise, the polypeptide can be identical to the avian Elf-1 polypeptide (chElf-1) shown in SEQ ID No: 4, or it can merely be homologous to that sequence. For instance, the polypeptide preferably has an amino acid sequence at least 70% homologous to the amino acid sequence in either or both of SEQ ID Nos: 2 and 4, though higher sequence homologies of, for example, 80%, 85%, 90% or 95% are also contemplated. The polypeptide can comprise the full length protein represented in SEQ ID No: 2 or 4, or it can comprise a fragment of that protein, which fragment may be, for instance, at least 5, 10, 20, 50 or 100 amino acids in length. A preferred Elf-1 polypeptide includes a Cys4 motif, such as the polypeptide between and including Cys-69 through Cys-159 of SEQ ID No: 2, or a sequence homologous thereto, such as residues 61-150 of SEQ ID No: 4 or residues 39-129 of SEQ ID No: 5. Yet another preferred Elf-1 polypeptide includes a core sequence motif, such as a polypeptide including residues 35-166 of SEQ ID No. 2, residues 33-157 of SEQ ID No. 4, or residues 5-136 of SEQ ID No. 5.
The polypeptide can be glycosylated, or, by virtue of the expression system in which it is produced, or by modification of the protein sequence to preclude glycosylation, reduced carbohydrate analogs can be provided. Likewise, Elf-1 polypeptides can be generated which lack an endogenous signal sequence (though this is typically cleaved off even if present in the pro-form of the protein), or which lack a phosphatidylinositol linkage site to preclude addition of phosphatidylinositol. In the instance of the latter, the removal of the C-terminus may result in a soluble form of the protein. In particular, polypeptides which lack at least the last 15 amino acid residues (the equivalent of muElf-1 truncated at Leu-195) are preferred, though polypeptides which are truncated anywhere between the equivalent of Thr-182 to Leu-195 of SEQ ID No: 2 are also contemplated.
Furthermore, the Elf-1 polypeptide can include a secretion signal sequence, such as residues Met-1 to Ala-20 of SEQ ID No. 2, though mature Elf-1 polypeptides are also provided, such as the exemplary full length mature polypeptide represented by residues Arg-21 to Ser-209.
Moreover, as described below, the polypeptide can be either an agonist (e.g. mimics), or alternatively, an antagonist of a biological activity of a naturally occuring form of the protein, e.g., the polypeptide is able to modulate growth and/or differentiation of a cell which expresses an EPH receptor.
In a preferred embodiment, a peptide having at least one biological activity of the subject polypepide may differ in amino acid sequence from the sequence in SEQ ID No: 2 or 4, but such differences result in a modified protein which functions in the same or similar manner as a native Elf-1 protein or which has the same or similar characteristics of a native Elf-1 protein. However, homologs of the naturally occuring protein are contemplated which are antagonistic of the normal physiological role of the naturally occurring protein. For example, the homolog may be capable of interfering with the ability of naturally-occurring forms of Elf-1 to modulate gene expression, e.g. of developmentally or growth regulated genes. Exemplary Elf-1 homologs are represented in the general formula:
In yet other preferred embodiments, the Elf-1 protein is a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated to Elf-1, e.g. the second polypeptide portion is glutathione-S-transferase, e.g. the second polypeptide portion is an enzymatic activity such as alkaline phosphatase, and is a reagent for detecting Elf-1 receptors.
Yet another aspect of the present invention concerns an immunogen comprising a Elf-1 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for an Elf-1 polypeptide; e.g. a humoral response, e.g. an antibody response; e.g. a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g. a unique determinant, from a protein represented by SEQ ID No. 2 or SEQ ID No. 4.
A still further aspect of the present invention features an antibody preparation specifically reactive with an epitope of the Elf-1 immunogen.
Another aspect of the present invention provides a substantially isolated nucleic acid having a nucleotide sequence which encodes an Elf-1 polypeptide. In preferred embodiments: the encoded polypeptide specifically binds an EPH receptor protein and/or is able to either agnoize or antagonize signal transduction events mediated by the EPH receptor. The coding sequence of the nucleic acid can comprise a sequence which can be identical to the cDNA shown in SEQ ID No: 1 or SEQ ID No: 3, or it can merely be homologous to that sequence. For instance, the Elf-1 encoding sequence preferably has a sequence at least 70% homologous to a nucleotide sequence of one or both of SEQ ID Nos: 1 and 3, though higher sequence homologies of, for example, 80%, 90% or 95% are also contemplated. The polypeptide encoded by the nucleic acid can comprise the amino acid sequence represented in SEQ ID No: 2 or 4, which is the full length protein, or it can comprise a fragment of that nucleic acid, which fragment may be, for instance, at least 5, 10, 20, 50 or 100 amino acids in length. The polypeptide encoded by the nucleic acid can be either an agonist (e.g. mimics), or alternatively, an antagonist of a biological activity of a naturally occuring form of the protein.
Furthermore, in certain preferred embodiments, the subject Elf-1 nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, which regulatory sequence is operably linked to the Elf-1 gene sequence. Such regulatory sequences can be used in to render the Elf-1 gene sequence suitable for use as an expression vector.
In a further preferred embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID No: 1; preferably to at least 20 consecutive nucleotides of SEQ ID No: 1; more preferably to at least 40 consecutive nucleotides of SEQ ID No: 1.
In yet a further preferred embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID No: 3; preferably to at least 20 consecutive nucleotides of SEQ ID No: 3; more preferably to at least 40 consecutive nucleotides of SEQ ID No: 3.
The invention also features transgenic non-human animals, e.g. mice, rats, rabbits, chickens, frogs or pigs, having a transgene, e.g., animals which include (and preferably express) a heterologous form of an Elf-1 gene described herein, or which misexpress an endogenous Elf-1 gene, e.g., an animal in which expression of the subject Elf-1 protein is disrupted. Such a transgenic animal can serve as an animal model for studying cellular and tissue disorders comprising mutated or mis-expressed Elf-1 alleles or for use in drug screening.
The invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence of SEQ ID No: 1 and/or 3, or naturally occurring mutants thereof. In preferred embodiments, the probe/primer further includes a label group attached thereto and able to be detected. The label group can be selected, e.g., from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors. Probes of the invention can be used as a part of a diagnostic test kit for identifying transformed cells, such as for detecting in a sample of cells isolated from a patient, a level of a nucleic acid encoding the subject Elf-1 proteins; e.g. measuring the Elf-1 mRNA level in a cell, or determining whether the genomic Elf-1 gene has been mutated or deleted. Preferably, the oligonucleotide is at least 10 nucleotides in length, though primers of, for example, 20, 30, 50, 100, or 150 nucleotides in length are also contemplated.
In yet another aspect, the invention provides an assay for screening test compounds for inhibitors, or alternatively, potentiators, of an interaction between Elf-1 and an EPH receptor. An exemplary method includes the steps of (i) combining an EPH receptor, an Elf-1 polypeptide, and a test compound, e.g., under conditions wherein, but for the test compound, the Elf-1 protein and the EPH receptor are able to interact; and (ii) detecting the formation of a complex which includes the Elf-1 protein and the receptor. A statistically significant change, such as a decrease, in the formation of the complex in the presence of a test compound (relative to what is seen in the absence of the test compound) is indicative of a modulation, e.g., inhibition, of the interaction between Elf-1 and the receptor. For example, primary screens are provided in which the Elf-1 protein and the receptor protein are combined in a cell-free system and contacted with the test compound; i.e. the cell-free system is selected from a group consisting of a cell lysate and a reconstituted protein mixture.
Another aspect of the present invention relates to a method of inducing and/or maintaining a differentiated state, causing proliferation, and/or enhancing survival of a cell responsive to a Elf-1 protein, by contacting the cells with an Elf-1 agonist or an Elf-1 antagonist. For example, the present method is applicable to cell culture technique, such as in the culturing of neuronal and other cells whose survival or differentiative state is dependent on Elf-1 function. Moreover, Elf-1 agonists and antagonists can be used for therapeutic intervention, such as to enhance survival and maintenance of neurons and other neural cells in both the central nervous system and the peripheral nervous system, as well as to influence other vertebrate organogenic pathways, such as other ectodermal patterning, as well as certain mesodermal and endodermal differentiation processes.
Another aspect of the present invention provides a method of determining if a subject, e.g. a human patient, is at risk for a disorder characterized by unwanted cell proliferation or abherent control of differentiation. The method includes detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding an Elf-1 protein, e.g. represented in SEQ ID No: 2 or 4, or a homolog thereof; or (ii) the mis-expression of an Elf-1 gene. In preferred embodiments, detecting the genetic lesion includes ascertaining the existence of at least one of: a deletion of one or more nucleotides from an Elf-1 gene; an addition of one or more nucleotides to the gene, a substitution of one or more nuclcotides of the gene, a gross chromosomal rearrangement of the gene; an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of the protein.
For example, detecting the genetic lesion can include (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of an Elf-1 gene, e.g. the nucleic acid represented in SEQ ID No: 1 or 3, or naturally occurring mutants thereof or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with the Elf-1 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e.g. wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the Elf-1 gene and, optionally, of the flanking nucleic acid sequences. For instance, the probe/primer can be employed in a polymerase chain reaction (PCR) or in a ligation chain reaction (LCR). In alternate embodiments, the level of Elf-1 protein is detected in an immunoassay using an antibody which is specifically immunoreactive with an Elf-1 protein.
Yet another aspect of the invention relates to a novel in situ assay for detecting receptors or their ligands in tissue samples and while organisms. In general, the xe2x80x9cRAP-in situxe2x80x9d assay (for Receptor Affinity Probe) of the present invention comprises (i) providing a hybrid molecule (the affinity probe) including a receptor, or a receptor ligand, covalently bonded to an enzymatically active tag, preferably for which chromogenic substrates exist, (ii) contacting the tissue or organism with the affinity probe to form complexes between the probe and a cognate receptor or ligand in the sample, removing unbound probe, and (iii) detecting the affinity complex using a chromogenic substrate for the enzymatic acitivity associated with the affinity probe. In preferred embodiments, an alkaline phosphatase provides an enzymatic tag, though such enzymes horseradish peroxidase, xcex2-galactosidase, malate dehydrogenase, yeast alcohol dehydrogenase, xcex1-glycerophosphate dehydrogenase, triose phosphate isomerase, asparaginase, glucose oxidase, and urease are also useful. The method can be used, for example, to detect patterns of expression for particular receptors and their ligands, for measuring the affinity of receptor/ligand interactions in tissue samples, as well as for generating drug screening assays in tissue samples. Moreover, the affinity probe can also be used in diagnostic screening to determine whether a receptor, e.g. an EPH receptor, or its ligand, e.g. Elf-1 or B61 or LERK proteins, are misexpressed.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.